The aims of the proposed research are to use 15N nuclear magnetic resonance (NMR) spectroscopy to investigate (1) the pathways of nitrogen metabolism in bacteria and fungi and (2) the mode of binding of an inhibitor, sulfanilamide, to carbonic anhydrase. The specific objectives for the metabolic experiements are (a) to investigate the relative contributions of two pathways of ammonia assimilation in Neurospora crassa and (b) to identify the intermediates and products of the pathways of arginine catabolism in Klebsiella aerogenes. We plan to accomplish these objectives by using 15N as a metabolic tracer and observing and identifying by 15N NMR spectroscopy the intermediates and products of the pathways in suspensions of intact cells and mycelia. The further development of in situ NMR has great potential as a clinical diagnostic tool since no perturbation of the biological system occurs. The specific objective for the enzymology experiments is to determine the precise mode of binding of the inhibitor, sulfanilamide, to the active-site zinc of carbonic anhydrase. Specifically, we plan to determine whether coordination is through the nitrogen atom of the inhibitor or through one of its oxygen atoms. We plan to obtain definitive evidence through measurement of the 15N chemical shift of the enzyme bound sulfanilamide. The results will be helpful in understanding the mechanisms of action of clinically important drugs. Our longterm objectives are to broaden the applicability of 15N NMR spectroscopy to the study of biological systems.